U.S. patent application number 10/343487 was filed with the patent office on 2004-02-26 for injectable protein formulations.
Invention is credited to Iida, Yoshimitsu, Tanikawa, Masahiko.
Application Number | 20040038878 10/343487 |
Document ID | / |
Family ID | 18729296 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038878 |
Kind Code |
A1 |
Tanikawa, Masahiko ; et
al. |
February 26, 2004 |
Injectable protein formulations
Abstract
An injectable pharmaceutical formulation containing a
physiologically active protein as an active ingredient and at least
one sugar as a soothing agent but containing no other proteins as
additives and having a pH of 6.5-7.4.
Inventors: |
Tanikawa, Masahiko; (Tokyo,
JP) ; Iida, Yoshimitsu; (Tokyo, JP) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Family ID: |
18729296 |
Appl. No.: |
10/343487 |
Filed: |
January 31, 2003 |
PCT Filed: |
August 6, 2001 |
PCT NO: |
PCT/JP01/06739 |
Current U.S.
Class: |
514/7.7 ;
424/130.1; 424/85.1; 514/18.3; 514/53; 514/738 |
Current CPC
Class: |
A61P 39/00 20180101;
A61K 47/26 20130101; A61K 38/00 20130101; A61P 7/00 20180101; A61K
9/0019 20130101; A61K 9/19 20130101; A61K 47/183 20130101 |
Class at
Publication: |
514/12 ; 514/53;
424/85.1; 424/130.1; 514/738 |
International
Class: |
A61K 038/19; A61K
038/23; A61K 031/7012; A61K 039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 2000 |
JP |
2000-237432 |
Claims
1. An injectable pharmaceutical formulation containing a
physiologically active protein as an active ingredient and at least
one sugar as a soothing agent but containing no other proteins as
additives and having a pH of 6.5-7.4.
2. The injectable formulation of claim 1 wherein the sugar is at
least one member selected from the group consisting of mannitol,
sorbitol, trehalose and sucrose.
3. The injectable formulation of claim 1 wherein the sugar is
mannitol.
4. The injectable formulation of any one of claims 1 to 3 which has
a pH of 6.8-7.2.
5. The injectable formulation of any one of claims 1 to 4 wherein
the physiologically active protein is erythropoietin, granulocyte
colony-stimulating factor or a monoclonal antibody.
6. The injectable formulation of claim 5 wherein the
physiologically active protein is erythropoietin.
7. The injectable formulation of claim 6 which is a yet to be
freeze-dried erythropoietin solution formulation.
8. The injectable formulation of any one of claims 1 to 7 which
further contains histidine and/or a salt thereof as a
stabilizer.
9. A method for reducing the pain caused by administration of
injectable formulations, comprising incorporating at least one
sugar into an injectable pharmaceutical composition and adjusting
the pH to 6.5-7.4.
10. The method of claim 9 wherein the sugar is mannitol.
Description
TECHNICAL FIELD
[0001] The present invention relates to injectable pharmaceutical
formulations containing a physiologically active protein as an
active ingredient and at least one sugar as a soothing agent but
containing no other proteins as additives and having a pH of
6.5-7.4. The present invention also relates to methods for reducing
the pain caused by administration of injectable formulations by
incorporating at least one sugar into an injectable pharmaceutical
composition and adjusting pH to 6.5-7.4.
BACKGROUND ART
[0002] Physiologically active proteins such as erythropoietin,
granulocyte colony-stimulating factor (G-CSF) and monoclonal
antibodies are unstable and susceptible to extrinsic factors such
as temperature, humidity, oxygen, UV rays or the like to undergo
physical or chemical changes such as denaturation, aggregation,
association, polymerization, oxidation, hydrolysis or disulfide
exchange reaction, resulting in great loss of activity.
[0003] A conventional means to control these chemical or physical
changes is to add proteins commonly used as stabilizers such as
human serum albumin. The addition of these proteins such as albumin
as stabilizers requires a complicated process to avoid the risk of
viral contamination or the like. However, we found that the problem
of pain arises from administration of injectable formulations
depending on the pH of the solution without adding such proteins as
albumin.
[0004] On the other hand, physiologically active proteins in
solution formulations are relatively more stable under acidic
conditions. For example, erythropoietin is known to be stable under
acidic conditions around pH 6.0.
[0005] However, such acidic conditions may cause pain from
administration of injections and therefore should be further
controlled.
[0006] An object of the present invention is to provide an
injectable protein formulation that will cause reduced pain from
administration and which has improved stability to allow long-term
storage.
[0007] Another object of the present invention is to provide a
method for reducing the pain caused by administration of injectable
formulations by incorporating at least one sugar into an injectable
pharmaceutical composition.
DISCLOSURE OF THE INVENTION
[0008] As a result of careful studies, we accomplished the present
invention on the basis of the findings that the pain caused by
administration is reduced when a sugar is included in place of
salts conventionally used as isotonizing agents such as sodium
chloride; the pain caused by administration is further reduced at a
pH of 6.5-7.4; and the formulations having a pH of 6.5-7.4 and
containing the sugar are also sufficiently improved in the
stability of a physiologically active protein as the principal
agent that they can be stored for an extended period of time.
[0009] The present invention provides an injectable formulation
containing a physiologically active protein as an active ingredient
and at least one sugar as a soothing agent but containing no other
proteins as additives and having a pH of 6.5-7.4 (hereinafter
referred to as "the formulation of the present invention" or "the
present formulation").
[0010] The present invention also provides a method for reducing
the pain caused by administration of injectable formulations
(hereinafter referred to as "the method of the present invention"
or "the present method"), comprising incorporating at least one
sugar into an injectable pharmaceutical composition (hereinafter
referred to as "injectable composition") and adjusting the pH to
6.5-7.4.
[0011] Preferred embodiments are as follows.
[0012] The present invention provides the injectable formulation as
defined above wherein the sugar is at least one member selected
from the group consisting of mannitol, sorbitol, trehalose and
sucrose.
[0013] The present invention provides the injectable formulation as
defined above wherein the sugar is mannitol.
[0014] The present invention provides the injectable formulation as
defined above which has a pH of 6.8-7.2.
[0015] The present invention provides the injectable formulation as
defined above wherein the physiologically active protein is
erythropoietin, granulocyte colony-stimulating factor (G-CSF) or a
monoclonal antibody.
[0016] The present invention provides the injectable formulation as
defined above wherein the physiologically active protein is
erythropoietin.
[0017] The present invention provides the injectable formulation as
defined above which further contains histidine and/or a salt
thereof as a stabilizer.
[0018] The present invention provides the method for reducing the
pain caused by administration of injectable formulations as defined
above wherein the sugar is mannitol.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows analytical results of the difference in pain
during injection of drug solutions, comparing the analytical
results from formulations B-E (upper panel), pHs 6 and 7 (middle
panel) and treatments with mannitol (M) and NaCl (N) (lower
panel).
[0020] FIG. 2 shows analytical results of the difference in pain
after injection of drug solutions, comparing the analytical results
from formulations B-E (upper panel), pHs 6 and 7 (middle panel) and
treatments with mannitol (M) and NaCl (N) (lower panel).
[0021] FIG. 3 shows analytical results of the pain scale during
injection of drug solutions, comparing the analytical results from
formulations A-E (upper panel), pHs 6 and 7 in contrast with
formulation A (middle panel) and treatments with mannitol (M) and
NaCl (N) in contrast to formulation A (lower panel).
[0022] FIG. 4 shows analytical results of the pain scale after
injection of drug solutions, comparing the analytical results from
formulations A-E (upper panel), pHs 6 and 7 in contrast to
formulation A (middle panel) and treatments with mannitol (M) and
NaCl (N) in contrast with formulation A (lower panel).
[0023] FIG. 5 shows analytical results of the difference from the
pain scale of the first injection during injection of drug
solutions, comparing the analytical results from formulations B-E
(upper panel), pHs 6 and 7 (middle panel) and treatments with
mannitol (M) and NaCl (N) (lower panel).
[0024] FIG. 6 shows analytical results of the difference from the
pain scale of the first injection after injection of drug
solutions, comparing the analytical results from formulations pHs 6
and 7 (upper panel), B-E (middle panel) and treatments with
mannitol (M) and NaCl (N) (lower panel).
THE MOST PREFERRED EMBODIMENTS OF THE INVENTION
[0025] As used herein, the "soothing agent" means an additive for
reducing the pain caused by administration of injections.
[0026] Sugars used as soothing agents in the present invention
include not only sugars in a narrow sense consisting of
monosaccharides, oligosaccharides and polysaccharides but also
sugar alcohols. Sugar alcohols here include not only linear
polyhydric alcohols obtained by reduction of carbonyl groups but
also cyclic alcohols.
[0027] Examples of these sugars include glucose, fructose,
saccharose, maltose, lactose, sucrose, mannose, raffinose,
mannitol, xylitol, galactitol, glucitol, inositol, sorbitol,
trehalose and glycerine. Mannitol, sorbitol, trehalose, sucrose,
inositol and glucose are preferably used, more preferably mannitol,
sorbitol, trehalose and sucrose, still more preferably
mannitol.
[0028] The amount of sugars used in the present invention is
determined such that the relative osmotic pressure of the present
formulation or the relative osmotic pressure of the injectable
composition as combined with a sugar in the present method can be
each controlled at about 0.5 to about 2.0, preferably about 1. The
"relative osmotic pressure" as used here is expressed as a relative
ratio based on the physiological osmotic pressure. That is, it
means a relative osmotic pressure as determined by calculation for
the concentration of sodium chloride, with the relative osmotic
pressure of 0.9 w/v % aqueous sodium chloride solution being taken
as 1.
[0029] Physiologically active proteins used as active ingredients
in the present invention include, but not limited to, hematopoietic
factors such as granulocyte colony-stimulating factor (G-CSF),
granulocyte macrophage colony-stimulating factor (GM-CSF),
erythropoietin (EPO) and thrombopoietin; cytokines such as
interferon, IL-1 and IL-6; monoclonal antibodies; tissue
plasminogen activator (TPA); urokinase; serum albumin; blood
coagulation factor VIII; leptin; insulin; and stem cell growth
factor (SCF). Preferred proteins are hematopoietic factors such as
EPO, G-CSF, GM-CSF and thrombopoietin and monoclonal antibodies,
more preferably EPO, G-CSF and monoclonal antibodies.
[0030] Physiologically active proteins used as active ingredients
in the present invention may be derived from natural sources or
preferably genetically engineered so far as they have substantially
the same biological activities as those of physiologically active
proteins of mammals, especially human. Genetically engineered
proteins may have the same amino acid sequences as those of natural
proteins or may contain deletion, substitution or addition of one
or more amino acids in the amino acid sequences while maintaining
the biological activities. Physiologically active proteins also
include those chemically modified with PEG or the like.
[0031] Physiologically active proteins used as active ingredients
in the present invention include, for example, proteins having a
sugar chain. The sugar chains may be those derived from any source,
but preferably those for glycosylation in mammalian cells.
Mammalian cells include, for example, Chinese hamster ovary (CHO)
cells, BHK cells, COS cells, human-derived cells, etc., among which
CHO cells are most preferred.
[0032] When the physiologically active protein used as an active
ingredient in the present invention is EPO, EPO may be prepared by
any process, e.g. it may be extracted from human urine and isolated
and purified by various techniques or may be produced by genetic
engineering techniques (see JP-A-61-012288, for example) in Chinese
hamster ovary (CHO) cells, BHK cells, COS cells, human-derived
cells or the like and then extracted and isolated and purified by
various techniques. EPO chemically modified with PEG or the like is
also included (see International Publication No. WO90/12874). EPO
that has no sugar chain and which has been chemically modified with
PEG or the like is also included. EPO analogs are also included, in
which EPO has been modified to increase the number of one or more
glycosylation sites at the N-linked carbohydrate chain binding site
or O-linked carbohydrate binding site in the amino acid sequence of
EPO (see JP-A-8-151398 and JP-A-8-506023, for example). Moreover,
the amount of sugar chains may be increased by increasing the
content of sialic acid or the like without changing the number of
sugar chain-binding sites.
[0033] When the physiologically active protein used as an active
ingredient in the present invention is G-CSF, any highly purified
G-CSF can be used. G-CSF in the present invention may be prepared
by any process, e.g., they may be extracted from cultures of a
human tumor cell line and isolated and purified by various
techniques or may be produced by genetic engineering techniques in
bacterial cells such as E. coli; yeast cells; animal culture cells
such as Chinese hamster ovary (CHO), C127 or COS cells and then
extracted and isolated and purified by various techniques. G-CSF is
preferably produced by genetic recombination in E. coli, yeast or
CHO cells, most preferably by genetic recombination in CHO cells.
G-CSF chemically modified with PEG or the like is also included
(see International Publication No. WO90/12874).
[0034] When the physiologically active protein used as an active
ingredient in the present invention is a monoclonal antibody, the
monoclonal antibody may be prepared by any process. Monoclonal
antibodies can be basically constructed by known techniques as
follows. A suitable host is immunized with an immunizing antigen
according to a standard immunization technique, and the resulting
immunized cells are fused to known parent cells by a standard cell
fusion technique, and then the fused cells are screened for
monoclonal antibody-producing cells by a standard screening method.
Monoclonal antibodies are not limited to those produced by
hybridomas, but also include chimeric antibodies obtained by
artificial modifications to lower heteroantigenicity to human or
for other purposes. Reshaped humanized antibodies can also be used
in the present invention, which are obtained by replacing the
complementarity-determining regions of a human antibody by the
complementarity-determining regions of a non-human mammalian
antibody such as a mouse antibody by standard gene recombination
techniques also known. These known techniques can be used to obtain
reshaped humanized antibodies useful in the present invention.
[0035] The protein formulation of the present invention or the
injectable composition used in the present method is free from
proteins as additives.
[0036] As used herein, the "proteins as additives" mean components
such as albumin and purified gelatin contained as stabilizers in
products currently supplied to the market to control chemical or
physical changes of protein formulations.
[0037] The present formulation and the injectable composition in
the present method may further contain stabilizers. One or more
stabilizers may be added in combination.
[0038] These stabilizers include amino acids such as histidine,
tryptophan, methionine, leucine, phenylalanine, serine, glutamic
acid, arginine, lysine and/or salts thereof, among which histidine
and/or salts thereof are preferably used.
[0039] The amount of stabilizers added to the protein formulation
of the present invention and the injectable composition in the
present method is typically 0.001-10% (w/v), preferably 0.01-1.0%
(w/v).
[0040] The formulation of the present invention and the injectable
composition in the present method may further contain surfactants.
One or more surfactants may be added in combination.
[0041] Preferred surfactants are polyoxyethylene sorbitan fatty
acid esters, more preferably Polysorbates 20, 21, 40, 60, 65, 80,
81, 85, most preferably Polysorbates 20 and 80.
[0042] The amount of surfactants added to the protein formulation
of the present invention and the injectable composition in the
present method is typically 0.00025-0.5% (w/v), preferably
0.001-0.1% (w/v).
[0043] The protein formulation of the present invention and the
injectable composition in the present method may further contain
diluents, solubilizing agents, pH-modifiers, buffers,
sulfur-containing reducing agents, antioxidants, preservatives or
the like, if desired. For example, preservatives include quaternary
ammonium salts such as benzalkonium chloride and benzethonium
chloride; and parabens such as methyl paraoxybenzoate, ethyl
paraoxybenzoate, propyl paraoxybenzoate and butyl paraoxybenzoate;
which may be used alone or in combination. Sulfur-containing
reducing agents include N-acetylcysteine, N-acetylhomocysteine,
thioctic acid, thiodiglycol, thioethanolamine, thioglycerol,
thiosorbitol, thioglycolic acid and salts thereof, sodium
thiosulfate, glutathione, and sulfhydryl-containing compounds such
as thioalkanoic acid having 1 to 7 carbon atoms. Antioxidants
include erythorbic acid, dibutylhydroxytoluene,
butylhydroxyanisole, .alpha.-tocopherol, tocopherol acetate,
L-ascorbic acid and salts thereof, L-ascorbyl palmitate, L-ascorbyl
stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl
gallate or chelating agents such as disodium ethylenediamine
tetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate.
Other components commonly added may also be contained, e.g.,
inorganic salts such as sodium chloride, potassium chloride,
calcium chloride, sodium phosphate, potassium phosphate, sodium
bicarbonate; and organic salts such as sodium citrate, potassium
citrate, sodium acetate.
[0044] Buffers used in the present formulation and the injectable
composition in the present method include acids commonly used as
buffers in injections and salts thereof or mixed solutions with a
base or a salt thereof, such as phosphoric acid, acetic acid,
hydrochloric acid, phthalic acid, boric acid, citric acid, carbonic
acid, succinic acid and salts thereof, preferably phosphate buffers
(sodium monohydrogen phosphate-sodium dihydrogen phosphate system)
and/or citrate buffers and/or acetate buffers.
[0045] The concentration of buffers used in the protein formulation
of the present invention and the injectable composition in the
present method is typically 0-300 mM, preferably 0-100 mM on the
basis of the total amount of the present formulation or the
injectable composition as combined with a sugar.
[0046] The pH of the present formulation and the injectable
composition as combined with a sugar in the present method is
preferably 6.5-7.4, more preferably 6.8-7.2.
[0047] The pH can be adjusted with commonly used pH-modifiers
including acids such as hydrochloric acid and bases such as sodium
hydroxide.
[0048] The protein formulation of the present invention or the
physiologically active protein used in the present method is in the
form of a solution, a freeze-dried or spray-dried formulation or
the like, most preferably a solution formulation.
[0049] The protein formulation of the present invention or the
injectable composition or physiologically active protein in the
present method is normally packed in a sealed and sterilized
plastic or glass container, for example. The container can be
supplied in the form having a defined volume such as ampules, vials
or disposable syringes or a large volume such as injection bags or
bottles. Freeze-dried formulations can be dissolved in pure water
(water for injection) before use.
[0050] Injections of the present invention include drip infusions
and are administered subcutaneously, intravenously or
intramuscularly, for example.
[0051] The concentration of physiologically active proteins used in
the present invention can be determined depending on the proteins
used, the type of disease to be treated, the severity of the
patient, the age of the patient and other factors. Generally,
proteins are contained in an amount of 0.5 .mu.g-100 mg/ml on the
basis of the total amount of the present formulation or the
injectable composition as combined with a sugar. For example, EPO
in a solution formulation is normally contained in an amount of
100-500,000 IU/ml (about 0.5-3000 .mu.g/ml), preferably 200-100,000
IU/ml (about 1-600 .mu.g/ml), more preferably 750-72,000 IU/ml
(about 4-400 .mu.g/ml). G-CSF is normally contained in an amount of
1-1000 .mu.g/ml, preferably 10-800 .mu.g/ml, more preferably 50-500
.mu.g/ml expressed as a final administration concentration. When
the physiologically active protein is an antibody such as
immunoglobulin, monoclonal antibodies and humanized antibodies, it
is contained in an amount of 0.1-200 mg/ml, preferably 1-200 mg/ml,
more preferably 10-200 mg/ml, most preferably 10-150 mg/ml
expressed as a final administration concentration.
[0052] The formulation of the present invention is improved in the
pain caused by administration and is very stable even after storage
at 25.degree. C. for 3 months, at 25.degree. C. for 6 months and at
40.degree. C. for 2 weeks, as shown in the following examples.
[0053] The following examples further illustrate the present
invention without, however, limiting the scope of the present
invention thereto. Various changes and modifications can be made by
those skilled in the art on the basis of the description of the
present invention, and such changes and modifications are also
included in the present invention.
EXAMPLES
Example 1
Pain Test
[0054] The difference in the pain caused by administration to human
of samples with varying hydrogen ion concentrations (pHs) and
isotonizing agents was examined in comparison with comparative
injections which contain sodium chloride as an isotonizing agent or
are adjusted to pH 6.
[0055] A. Test Method
[0056] Test Drugs:
[0057] A comparative drug (drug A) and the following samples B-E
with varying pHs and isotonizing agents (with the pH and
composition of each sample shown in Table 1) were prepared. All the
drugs were prepared with the same buffer (phosphate) and the same
relative osmotic pressure (1.0), and contain about 10 mmol
phosphate, about 2.5 mmol NaCl or about 250 mmol mannitol, 0.1%
L-histidine as a stabilizer and 0.005% Polysorbate 80 as an
adsorption inhibitor.
1TABLE 1 List of samples administered Relative osmotic Drug
Formulation details pressure A pH 6.0, phosphate, Tween 80, 1.0
histidine, NaCl B pH 6.3, phosphate, Tween 80, 1.0 histidine, NaCl
C pH 7.0, phosphate, Tween 80, 1.0 histidine, NaCl D pH 7.0,
phosphate, Tween 80, 1.0 histidine, mannitol E pH 6.0, phosphate,
Tween 80, 1.0 histidine, mannitol
[0058] Administration Method of Test Drugs:
[0059] The samples above (Table 1) were paired according to a
combination table (Table 2) and administered to the upper arm of
each subject (18 normals) at an interval (about 30 seconds) using a
syringe containing a predetermined amount (0.5 mL) of each sample
(First injection: drug A presumed to cause the strongest pain (a
sample of the prior art); Second injection: a sequence of samples
according to the combination. table). The difference in pain as
compared with the first injection and the severity of pain of the
first and second injections were recorded.
2TABLE 2 Combination table for pain test Subject No. First
injection Second injection 1 A B 2 A C 3 A B 4 A D 5 A C 6 A D 7 A
B 8 A C 9 A B 10 A D 11 A C 12 A D 13 A B 14 A E 15 A C 16 A E 17 A
D 18 A E
[0060] Evaluation Items and Evaluation Criteria:
[0061] The following three items were evaluated during and after
injection of drug solutions.
[0062] 1. Difference in Pain
[0063] The severity of the pain caused by the second injection was
assessed at 9 scales depending on whether it increased or decreased
from the severity of the pain caused by the first injection (drug
A) so that in total the severity of pain was assessed at 19 scales;
the difference in pain between the first and second injections was
recorded under the scale bar at the corresponding scale (Table
3).
3TABLE 3 1) During injection of drug solutions Milder (-) Less More
pain than in Pain than in the First the first injection first
injection injection Severer (+) 9 8 7 6 5 4 3 2 1 0 1 2 3 4 5 6 7 8
9 Second injection 2) After injection of drug solutions Milder (-)
Less More pain than in Pain than in the First the first injection
first injection injection Severer (+) 9 8 7 6 5 4 3 2 1 0 1 2 3 4 5
6 7 8 9 Second injection
[0064] 2. Pain Scale
[0065] At each of the first and second administrations, the
severity of pain was assessed by the following six criteria and
recorded. In the 6-score assessment, the cases of severity shown in
Table 4 were rated by numerals 0-5 as follows: "no pain", 0; "very
mild", 1; "mild", 2; "moderate", 3; "severe", 4; and "unbearable",
5.
[0066] The evaluation item of "pain scale" was based on a standard
wherein smaller values indicate lower severity of pain. Thus, the
smaller the value in each evaluation item, the less the pain caused
by the drug solution.
4 TABLE 4 First injection Second injection 1) During injection of
drug solutions No pain No pain Very mild Very mild Mild Mild
Moderate Moderate Severe Severe Unbearable Unbearable 2) After
injection of drug solutions No pain No pain Very mild Very mild
Mild Mild Moderate Moderate Severe Severe Unbearable Unbearable
[0067] 3. Difference from the Pain Scale of the First Injection
(Drug A)
[0068] The difference in evaluation 2 between the first (drug A)
and second injections.
[0069] B. Analytical Method
[0070] [1] Analysis of variance was performed on each evaluation
item during and after injection of drug solutions.
[0071] [2] Analysis of variance was performed using pH 6.0 for
drugs B and E (B was supposed to be at pH 6.0 for analysis) and pH
7.0 for drugs C and D as variant [pH] as well as NaCl for drugs B
and C and mannitol for drugs D and E as variant [ISO]. In the
analysis of "pain scale", drugs A to E were not totally analyzed
but drugs B to E were compared. For drug A, however, the
average.+-.standard error was shown as reference in the graphs
without being compared with the other drugs.
[0072] C. Results
[0073] 1. Difference in Pain
[0074] Analytical results of the difference in pain during and
after injection of drug solutions are shown in FIGS. 1 and 2,
respectively.
[0075] 1.1 During Injection of Drug Solutions
[0076] [1] The effect of the drug (the main effect of the drug) was
studied to show that the severity of the pain caused by drugs B to
E was in the order of D<C<B.apprxeq.E with drug D being
improved by about 5 levels on the scale of difference in pain as
compared with drug A
[0077] [2] The effect of the pH of the drug solution was studied to
show that the pain was less at pH 7 than pH 6 with solutions at pH
7 being improved by about 4 levels on the scale of difference in
pain as compared with drug A.
[0078] [3] The effect of the isotonizing agent was studied at each
stage to show that mannitol caused less pain than NaCl.
[0079] 1.2 After Injection of Drug Solutions
[0080] [1] The effect of the drug (the main effect of the drug) was
studied to show that the severity of the pain caused by drugs B to
E was in the order of D<B.apprxeq.C.apprxeq.E with drug D being
improved by about 3 levels on the scale of difference in pain as
compared with drug A.
[0081] [2] The effect of the pH of the drug solution was studied to
show that the pain was less at pH 7 than pH 6.
[0082] [3] The effect of the isotonizing agent was studied to show
that both isotonizing agents brought improvement by about 2 levels
on the scale of difference in pain as compared with drug A.
[0083] 2. Pain Scale
[0084] Analytical results of the pain scale during and after
injection of drug solutions are shown in FIGS. 3 and 4,
respectively.
[0085] 2.1 During Injection of Drug Solutions
[0086] [1] The effect of the drug was studied to show that the
severity of pain was in the order of D<C=E<B with drug D
being improved by about 2 levels on the "pain scale" as compared
with drug A. Test results showed that drug D was significantly
different from any other drugs.
[0087] [2] The effect of the pH of the drug solution was studied to
show that the pain was less at pH 7 than pH 6 with solutions at pH
7 being improved by about 1 level on the "pain scale" as compared
with drug A.
[0088] [3] The effect of the isotonizing agent was studied to show
that mannitol caused significantly less pain than NaCl.
[0089] 2.2 After Injection of Drug Solutions
[0090] [1] The effect of the drug was studied to show that all the
drugs were nearly comparable at about 1.5 on the pain scale with D
showing a somewhat lower value.
[0091] [2] The effect of the pH of the drug solution was studied to
show a smaller value at pH 7 than pH 6.
[0092] [3] The effect of the isotonizing agent was studied to show
that mannitol and NaCl showed nearly comparable values of about 1.5
on the pain scale.
[0093] 3. Difference in Pain Scale
[0094] Analytical results of the difference in pain scale between
the first (drug A) and second injections during and after injection
of drug solutions are shown in FIGS. 5 and 6, respectively.
[0095] 3.1 During Injection of Drug Solutions
[0096] [1] The effect of the drug was studied to show that the
severity of pain was in the order of D<C<E<B.
[0097] [2] The effect of the pH of the drug solution was studied to
show that the pain was significantly less at pH 7 than pH 6,
similarly to the "difference in pain".
[0098] [3] The effect of the isotonizing agent was studied to show
that mannitol caused significantly less pain than NaCl.
[0099] 3.2 After Injection of Drug Solutions
[0100] [1] The effect of the drug was studied to show a difference
in pain scale of -1 or less in each of the drugs.
[0101] [2] The effect of the pH of the drug solution was studied to
show that the results were nearly comparable at both pHs with the
difference in "pain scale" being less than -1.
[0102] [3] The effect of the isotonizing agent was studied to show
that the effects of mannitol and NaCl were nearly comparable with
the difference in "pain scale" being less than -1.
[0103] E. Discussion
[0104] 1) As to the influence of the hydrogen ion concentration
(pH) on the pain during injection of drug solutions, the severity
of pain was clearly lower at pH 7.0 than pH 6.0.
[0105] 2) As to the influence of the isotonizing agent on the pain
during injection of drug solutions, mannitol was excellent.
[0106] 3) Formulation D was the most excellent in the pain after
injection of drug solutions within the range of this test, but all
the formulations including formulation A caused mild pain with no
practical problem.
Example 2
Stability Test
[0107] Erythropoietin solution formulations with varying
formulation factors found to influence pain in Example 1 (pH and/or
isotonizing agent) were prepared as test samples and evaluated for
stability by quantification and purity assays.
[0108] Specifically, stability was compared between formulations
containing D-mannitol (hereinafter referred to as "Man added") and
formulations containing sodium chloride (hereinafter referred to as
"Man not added") because the result of the pain test on normals in
Example 1 showed that the pain caused by injection of drug
solutions was relieved when D-mannitol was used as an isotonizing
agent as compared with sodium chloride. Stability of erythropoietin
solutions under weak acid to weak alkaline conditions was also
examined because the pain was relieved at pH 7.0 as compared with
pH 6.0.
[0109] A. Test Samples
[0110] All the samples used in formulation studies contain 750 IU
erythropoietin in 0.5 mL. The samples tested are shown in Table 5.
All the samples used were prepared as erythropoietin (genetically
engineered EPO) solution formulations (1500 IU/ml) containing
L-histidine (0.1%) as a stabilizer and Polysorbate 80 (0.005%)
using the same buffer (phosphate) and the same relative osmotic
pressure (1.0). They contain about 10 mmol phosphate and about 2.5
mmol NaCl or about 250 mmol D-mannitol.
5TABLE 5 Components of test samples pH, buffer, adsorption
inhibitor, Formulation stabilizer, isotonizing agent A pH 6.0,
phosphate, Tween 80, histidine, NaCl B pH 6.5, phosphate, Tween 80,
histidine, NaCl C pH 7.0, phosphate, Tween 80, histidine, NaCl D pH
7.5, phosphate, Tween 80, histidine, NaCl E pH 6.0, phosphate,
Tween 80, histidine, mannitol F pH 7.0, phosphate, Tween 80,
histidine, mannitol
[0111] B. Test Method
[0112] Each formulation test sample was stored at 25.degree. C. for
6 months and evaluated by purity assay 1 [product-related
impurities: aggregates (SDS-PAGE)], purity assay 2 [product-related
impurities: low molecular weight degradation products (SDS-PAGE(R)]
and quantification [liquid chromatography (HPLC)].
[0113] 1. Purity Assay 1 [Product-Related Impurities: Aggregates
(SDS-PAGE)]
[0114] (1) Test Method
[0115] Each sample solution, a standard solution and a molecular
weight marker solution were electrophoresed and detected by Western
blotting.
[0116] 2. Purity Assay 2 [Product-Related Impurities: Low Molecular
Weight Degradation Products (SDS-PAGE(R)]
[0117] (1) Test Method
[0118] A 7.5 .mu.L aliquot of each sample was electrophoresed and
detected by Western blotting using an anti-erythropoietin antibody
(prepared as a polyclonal antibody).
[0119] The band detected around 32 kd was read with a densitometer
to calculate the concentration from the staining strength of the
sample.
[0120] 3. Quantification [Liquid Chromatography (HPLC)]
[0121] (1) Test Method
[0122] Liquid chromatography was used to determine the content of
erythropoietin (% of the nominal potency) from the peak area of
erythropoietin.
[0123] 4. Discussion and/or Conclusion
[0124] [Influence of pH on the Stability of Erythropoietin
Solutions]
[0125] Table 6 shows the relation between pH and the residual rate
by HPLC of erythropoietin solutions (1500 IU/mL) after storage at
25.degree. C. for 6 months (the results of formulations A-D).
Taking into account of variation in measurement results, the
residual rate by HPLC remained unchanged at pH 6.0-7.0 and
decreased at pH 7.5.
6TABLE 6 Influence of pH on the stability of erythropoietin
solutions (1500 IU/mL) pH 6.0 6.5 7.0 7.5 Residual rate (%) by HPLC
94.9 93.3 92.4 89.3 after storage at 25.degree. C. for 6 months
[0126] [Influence of Sugar on the Stability of Erythropoietin
Solutions]
[0127] Table 7 shows the relation between Man addition and the
residual rate of erythropoietin solutions (1500 IU/mL) by HPLC
after storage at 25.degree. C. for 6 months (the results of
formulations E, F compared with formulations A, C). Although any
formulations at both pH 6.0 and pH 7.0 had no problem with
stability, the formulation containing no Man at pH 6.0 and the
formulation containing Man at pH 7.0 showed somewhat higher
contents by HPLC.
7TABLE 7 Influence of sugar on the stability of erythropoietin
solutions (1500 IU/mL) Residual rate (%) by HPLC after storage at
25.degree. C. for 6 months pH 6.0 pH 7.0 Man not added 94.9 92.4
Man added 93.3 94.0
CONCLUSION
[0128] The foregoing results show that formulation F, which is an
erythropoietin solution formulation having a pH of 7.0 and
containing D-mannitol as an isotonizing agent, is the most
preferred in the pain reduction caused by injection and the
stability of the formulation.
[0129] Comparison of the stability of formulations F and A after
storage at 25.degree. C. for 6 months revealed that formulation F
was comparable to formulation A in both residual rate and
degradation species.
[0130] It was concluded from this result that formulation F should
be stably stored at 10.degree. C. for 2 years or more as in the
case of formulation A which is known to be stable at 10.degree. C.
for 2 years or more.
* * * * *